白色念珠菌致病力相關基因STU2及PMC1之遺傳學研究

Abstract

白色念珠菌為一種伺機性病原菌，在健康人體內為正常共生菌。近年來隨著免疫不全病人之人數增加、不當使用抗生素，使得黴菌感染率顯著增加。EFG1及CPH1雙基因突變株(cph1/cph1 efg1/efg1)會抑制菌絲生長，且致病能力下降。酵母菌型與菌絲型之間的型態轉變對致病力之影響是重要的。之前實驗室利用抑制刪除雜交法(SSH)技術篩選影響白色念珠菌菌絲生長及抗致病力之基因。本論文之目的在於利用北方點墨法應證SSH之結果，並利用基因破壞方式研究與致病力相關之基因功能。共選擇九個基因以北方點墨法分析基因表現量。結果顯示其中五個基因在EFG1及CPH1雙基因突變株(cph1/cph1 efg1/efg1)之mRNA表現量大於野生株(CPH1/CPH1 EFG1/EFG1)，符合SSH之結果。三個基因在EFG1及CPH1雙基因突變株(cph1/cph1 efg1/efg1)之mRNA表現量與野生株(CPH1/CPH1 EFG1/EFG1)之間差異不大，一個基因在EFG1及CPH1雙基因突變株(cph1/cph1 efg1/efg1)之mRNA表現量小於野生株(CPH1/CPH1 EFG1/EFG1)不符合SSH之結果。選擇STU2與PMC1利用重組置換達到基因破壞之目的。所得到之菌株以PCR及南方點墨法確認是否正確。利用四環黴素調控表現系統將STU2與PMC1進行過度表現。已建構含有TR promoter之STU2與PMC1過量表現菌株，可將之應用於調控基因表現上之研究。

Candida albicans is an opportunistic pathogen that usually lives as a commensal in the healthy human host. With an increase in the number of immunocompromised patients and the abuse of antibiotics, the incidence of fungal infections has risen dramatically in recent years. The cph1/cph1 efg1/efg1 double mutant is defective in filamentous and avirulent in a mouse model. The yeast-hypha morphological transition is thought to be an important contributor to virulence. Previously researchers in the laboratory have used the method of suppressive subtractive hybridization to screen the antivirulence-association genes of Candida albicans. These genes may affect the hyphal growth and virulence. The aims of this research were to assess the results of suppressive subtractive hybridization by Northern blot and study the gene function associated with virulence by genetic knockout and overexpression. Nine genes were chosen for Northern blot analysis. The results showed that five of them had much higher mRNA expression level in cph1/cph1 efg1/efg1 double mutant than that of wild type, which were consistent with the results of suppressive subtractive hybridization. And three genes showed no difference between double mutant and wild type. One gene showed much higher mRNA expression level in wild type than in cph1/cph1 efg1/efg1 double mutant. The association between the morphogenesis and gene function were studied on two of those genes. STU2 and PMC1 genes were subjected to genetic knockouted by homologous recombination. Knockout clones were assessed by PCR and Southern blot. I also overexpressed STU2 and PMC1 genes in Candida albicans by the tetracycline-regulatable system. These strains were useful in gene regulation.